TLR-induced PAI-2 expression suppresses IL-1 processing ... · TLR-induced PAI-2 expression...

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TLR-induced PAI-2 expression suppresses IL-1β processing via increasing autophagy and NLRP3 degradation Shih-Yi Chuang a , Chih-Hsiang Yang a , Chih-Chang Chou a , Yu-Ping Chiang b , Tsung-Hsien Chuang c , and Li-Chung Hsu a,1 a Institute of Molecular Medicine, National Taiwan University, Taipei 10002, Taiwan; b Department of Pediatrics, National Taiwan University Hospital, Taipei 10002, Taiwan; and c Immunology Research Center, National Health Research Institutes, Miaoli County 35053, Taiwan Edited by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved August 15, 2013 (received for review April 11, 2013) The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inammasome, a multiprotein complex, triggers caspase-1 activa- tion and maturation of the proinammatory cytokines IL-1β and IL-18 upon sensing a wide range of pathogen- and damage-asso- ciated molecules. Dysregulation of NLRP3 inammasome activity contributes to the pathogenesis of many diseases, but its regula- tion remains poorly dened. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck- like protein containing a C-terminal caspase recruitment domain)dependent caspase-1 activation and IL-1β secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabi- lized the autophagic protein Beclin 1 to promote autophagy, result- ing in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and proIL-1β processing. Likewise, overexpressing Beclin 1 in PAI-2decient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inammasome activation and re- veal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1βdriven inammation. I nnate immunity, the rst line of host defense against pathogen infection, is composed of diverse germ line-encoded pattern- recognition receptors, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), that recognize pathogen-associated molecular patterns (PAMPs) from pathogens or danger-associ- ated molecular patterns from damaged tissue (1, 2). TLRs rec- ognize a variety of PAMPs from microbes to induce autophagy and cytokine production for host defense against microbial infections. Inammasomes, multiple protein complexes contain- ing NLR proteins or AIM2, mediate caspase-1 activation leading to the processing of the proinammatory cytokines IL-1β and IL- 18 (3). The inammasome/caspase-1 complexes also may target additional effector molecules to regulate diverse physiological functions, such as pyroptosis and tissue repair (4). Among the identied inammasomes, the NLRP3 inammasome has been studied extensively and has been shown to be activated by a large variety of activators that share no structural similarity (2). For this reason, it has been suggested that the NLRP3 inammasome is activated through a secondary mediator, such as potassium (K + ) efux, reactive oxygen species (ROS), or lysosomal proteases (1). The inammasomes play a critical role in the clearance of mi- crobial pathogens and tissue repair (2, 5). However, dysregulation of inammasome activation has been associated with a variety of human diseases, including autoinammatory diseases, metabolic disorders, and cancer (3, 6). Autophagy, an evolutionarily conserved cellular catabolic process, facilitates the recycling of damaged proteins and organ- elles (7). Increasing evidence indicates that autophagy is involved in the regulation of immune responses and inammation (7). Macrophages treated with an autophagy inhibitor or with the deletion of several autophagic components, including Atg16L1, Beclin 1, and LC3, induced greater caspase-1 activation and IL- 1β secretion in response to LPS or LPS plus an NLRP3 agonist (8, 9). These data strongly suggest that the NLRP3 inamma- some activity is negatively regulated by autophagy, but the un- derlying mechanism is poorly understood. Plasminogen activator inhibitor type 2 (PAI-2), a serine pro- teinase inhibitor (SERPIN), originally was identied as an inhibitor of the urokinase-type plasminogen activator (uPA) involved in cellular invasion and tissue remodeling (10). Recently, PAI-2 has been associated with newly identied uPA-independent biological functions, probably through targeting an as yet uncharacterized intracellular molecule (11). In addition, PAI-2 is one of the major molecules up-regulated in macrophages in response to TLR4 activators or inammatory mediators, suggesting its function in the regulation of innate immunity (12, 13). Macrophages treated with LPS alone do not release mature IL-1β and IL-18 unless accompanied by a second stimulus, such as ATP or crystals (8, 14). LPS activates TLR4 to induce the synthesis of proIL-1β and the inammasome component NLRP3 via IκB kinase (IKK)/NF-κB activation; a second stimulus is re- quired for inammasome assembly and caspase-1 activation to cleave proIL-1β and proIL-18 to their mature forms. Never- theless, previous work showed that LPS alone is sufcient to in- duce mature IL-1β production in IKKβ-decient macrophages because of enhanced proIL-1β processing (15). Additionally, LPS-induced PAI-2 expression is blunted in IKKβ-decient mac- rophages, and reintroduction of PAI-2 blocks IL-1β maturation in a caspase-1dependent manner, suggesting that PAI-2 inhibits proIL-1β processing upon LPS stimulation; however, the un- derlying mechanism is unknown. Here, we show that depletion of PAI-2 in macrophages induces caspase-1 activation and IL-1β production in response to TLR agonists and Escherichia coli with no need of a second stimulus. TLR engagement induced PAI-2 expression and en- hanced association of PAI-2 with Beclin 1, leading to an increase in autophagy, which then caused reduced mitochondrial ROS (mROS) and increased NLRP3 degradation, resulting in de- creased IL-1β maturation. Inammatory cytokines and cellular ROS play vital roles in innate immunity, but prolonged and excess production of these mediators can be detrimental. Our results suggest that PAI-2 is a cell-autonomous mechanism that counteracts the detrimental effects caused by TLR2/4- and E. coli-triggered cellular stress by reducing ROS production and the Author contributions: S.-Y.C., C.-H.Y., and L.-C.H. designed research; S.-Y.C., C.-H.Y., C.-C.C., and Y.-P.C. performed research; T.-H.C. contributed new reagents/analytic tools; S.-Y.C., C.-H.Y., Y.-P.C., and L.-C.H. analyzed data; and S.-Y.C., T.-H.C., and L.-C.H. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1306556110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1306556110 PNAS | October 1, 2013 | vol. 110 | no. 40 | 1607916084 IMMUNOLOGY

Transcript of TLR-induced PAI-2 expression suppresses IL-1 processing ... · TLR-induced PAI-2 expression...

TLR-induced PAI-2 expression suppresses IL-1βprocessing via increasing autophagy andNLRP3 degradationShih-Yi Chuanga, Chih-Hsiang Yanga, Chih-Chang Choua, Yu-Ping Chiangb, Tsung-Hsien Chuangc, and Li-Chung Hsua,1

aInstitute of Molecular Medicine, National Taiwan University, Taipei 10002, Taiwan; bDepartment of Pediatrics, National Taiwan University Hospital, Taipei10002, Taiwan; and cImmunology Research Center, National Health Research Institutes, Miaoli County 35053, Taiwan

Edited by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved August 15, 2013 (received for reviewApril 11, 2013)

The NOD-like receptor family, pyrin domain containing 3 (NLRP3)inflammasome, a multiprotein complex, triggers caspase-1 activa-tion and maturation of the proinflammatory cytokines IL-1β andIL-18 upon sensing a wide range of pathogen- and damage-asso-ciated molecules. Dysregulation of NLRP3 inflammasome activitycontributes to the pathogenesis of many diseases, but its regula-tion remains poorly defined. Here we show that depletion ofplasminogen activator inhibitor type 2 (PAI-2), a serine proteaseinhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)‐dependent caspase-1 activation and IL-1β secretion in macrophagesupon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 orTLR4 agonist induced PAI-2 expression, which subsequently stabi-lized the autophagic protein Beclin 1 to promote autophagy, result-ing in decreases in mitochondrial reactive oxygen species, NLRP3protein level, and pro–IL-1β processing. Likewise, overexpressingBeclin 1 in PAI-2–deficient cells rescued the suppression of NLRP3activation in response to LPS. Together, our data identify a tier ofTLR signaling in controlling NLRP3 inflammasome activation and re-veal a cell-autonomous mechanism which inversely regulates TLR- orEscherichia coli-induced mitochondrial dysfunction, oxidative stress,and IL-1β–driven inflammation.

Innate immunity, the first line of host defense against pathogeninfection, is composed of diverse germ line-encoded pattern-

recognition receptors, such as Toll-like receptors (TLRs) andNOD-like receptors (NLRs), that recognize pathogen-associatedmolecular patterns (PAMPs) from pathogens or danger-associ-ated molecular patterns from damaged tissue (1, 2). TLRs rec-ognize a variety of PAMPs from microbes to induce autophagyand cytokine production for host defense against microbialinfections. Inflammasomes, multiple protein complexes contain-ing NLR proteins or AIM2, mediate caspase-1 activation leadingto the processing of the proinflammatory cytokines IL-1β and IL-18 (3). The inflammasome/caspase-1 complexes also may targetadditional effector molecules to regulate diverse physiologicalfunctions, such as pyroptosis and tissue repair (4). Among theidentified inflammasomes, the NLRP3 inflammasome has beenstudied extensively and has been shown to be activated by a largevariety of activators that share no structural similarity (2). For thisreason, it has been suggested that the NLRP3 inflammasome isactivated through a secondary mediator, such as potassium (K+)efflux, reactive oxygen species (ROS), or lysosomal proteases (1).The inflammasomes play a critical role in the clearance of mi-crobial pathogens and tissue repair (2, 5). However, dysregulationof inflammasome activation has been associated with a variety ofhuman diseases, including autoinflammatory diseases, metabolicdisorders, and cancer (3, 6).Autophagy, an evolutionarily conserved cellular catabolic

process, facilitates the recycling of damaged proteins and organ-elles (7). Increasing evidence indicates that autophagy is involvedin the regulation of immune responses and inflammation (7).Macrophages treated with an autophagy inhibitor or with the

deletion of several autophagic components, including Atg16L1,Beclin 1, and LC3, induced greater caspase-1 activation and IL-1β secretion in response to LPS or LPS plus an NLRP3 agonist(8, 9). These data strongly suggest that the NLRP3 inflamma-some activity is negatively regulated by autophagy, but the un-derlying mechanism is poorly understood.Plasminogen activator inhibitor type 2 (PAI-2), a serine pro-

teinase inhibitor (SERPIN), originally was identified as an inhibitorof the urokinase-type plasminogen activator (uPA) involved incellular invasion and tissue remodeling (10). Recently, PAI-2 hasbeen associated with newly identified uPA-independent biologicalfunctions, probably through targeting an as yet uncharacterizedintracellular molecule (11). In addition, PAI-2 is one of the majormolecules up-regulated in macrophages in response to TLR4activators or inflammatory mediators, suggesting its function in theregulation of innate immunity (12, 13).Macrophages treated with LPS alone do not release mature

IL-1β and IL-18 unless accompanied by a second stimulus, suchas ATP or crystals (8, 14). LPS activates TLR4 to induce thesynthesis of pro–IL-1β and the inflammasome component NLRP3via IκB kinase (IKK)/NF-κB activation; a second stimulus is re-quired for inflammasome assembly and caspase-1 activation tocleave pro–IL-1β and pro–IL-18 to their mature forms. Never-theless, previous work showed that LPS alone is sufficient to in-duce mature IL-1β production in IKKβ-deficient macrophagesbecause of enhanced pro–IL-1β processing (15). Additionally,LPS-induced PAI-2 expression is blunted in IKKβ-deficient mac-rophages, and reintroduction of PAI-2 blocks IL-1β maturation ina caspase-1–dependent manner, suggesting that PAI-2 inhibitspro–IL-1β processing upon LPS stimulation; however, the un-derlying mechanism is unknown.Here, we show that depletion of PAI-2 in macrophages

induces caspase-1 activation and IL-1β production in response toTLR agonists and Escherichia coli with no need of a secondstimulus. TLR engagement induced PAI-2 expression and en-hanced association of PAI-2 with Beclin 1, leading to an increasein autophagy, which then caused reduced mitochondrial ROS(mROS) and increased NLRP3 degradation, resulting in de-creased IL-1β maturation. Inflammatory cytokines and cellularROS play vital roles in innate immunity, but prolonged andexcess production of these mediators can be detrimental. Ourresults suggest that PAI-2 is a cell-autonomous mechanism thatcounteracts the detrimental effects caused by TLR2/4- and E.coli-triggered cellular stress by reducing ROS production and the

Author contributions: S.-Y.C., C.-H.Y., and L.-C.H. designed research; S.-Y.C., C.-H.Y.,C.-C.C., and Y.-P.C. performed research; T.-H.C. contributed new reagents/analytictools; S.-Y.C., C.-H.Y., Y.-P.C., and L.-C.H. analyzed data; and S.-Y.C., T.-H.C., and L.-C.H.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1306556110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306556110 PNAS | October 1, 2013 | vol. 110 | no. 40 | 16079–16084

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inflammasome activation, thereby resulting in less inflammationand tissue damage.

ResultsLoss of PAI-2 Enhances LPS-Induced IL-1β Production and Caspase-1Activation. To investigate the function of PAI-2 in regulatingLPS-induced IL-1β production, we used human myelomonocyticTHP-1 cell-derived macrophages, which do not express PAI-2protein (16), as a model. In line with previous reports, LPStreatment alone induced IL-1β secretion in these cells (Fig. 1A)(14, 17). Other than LPS, treatment with tripalmitoyl cysteinyllipopeptide (Pam3CSK4; TLR2 ligand), but not with poly-inosinic-polycytidylic acid [poly(I:C); TLR3 ligand] or CpG(TLR9 ligand), also activated IL-1β processing (Fig. S1A). Pre-treatment with polymyxin B, an antibiotic that neutralizes LPSfunction, completely abrogated both IL-1β and TNF maturationin response to LPS but had no effect on Pam3CSK4-inducedcytokine production (Fig. S1B), thus indicating ligand specificityin the pathways leading to IL-1β production. Reintroduction ofPAI-2 into these cells diminished LPS- and Pam3CSK4-inducedIL-1β and IL-18 but had no effect on TNF production (Fig. 1Aand Fig. S1C). In addition, caspase-1 activation was suppressedin PAI-2–expressing THP-1 macrophages. Interestingly, PAI-2mRNA expression in macrophages was induced significantlyby LPS and Pam3CSK4 but not by poly(I:C) or CpG (Fig. S1D).We also depleted PAI-2 expression in human U937-derivedmacrophages, in which the PAI-2 gene product is functional, andin mouse bone marrow-derived macrophages (BMDMs) bylentivirus-mediated shRNA. Stimulation of U937 macrophagesand BMDMs expressing control shRNA (shLuc) with LPS alonedid not induce mature IL-1β and active caspase-1 (p20) pro-duction, whereas depletion of PAI-2 in these cells resulted incaspase-1 activation and IL-1β secretion in response to LPS (Fig.

1 B and C). LPS treatment did not cause obvious cell death, asjudged by undetectable lactate dehydrogenase (LDH) release inculture medium, ruling out the possibility that LPS-induced IL-1β release in PAI-2–deficient cells was the consequence of celllysis (Fig. S1E).PAI-2 expression did not affect activation of IKKβ and MAP

kinases (Fig. S2A), mRNA expression of NLRP3, IL-1β, andTNF (Fig. S2B), or protein levels of pro–IL-1β, procaspase-1,and apoptosis-associated Speck-like protein containing a C-terminalcaspase recruitment domain (ASC) after LPS treatment (Fig. 1 Aand C). We therefore examined whether PAI-2 modulates pro–IL-1β processing in response to LPS. Enhanced IL-1β secretionby LPS was markedly blocked by either a pan-caspase inhibitor(zVAD-fluoromethylketone) or a caspase-1 inhibitor (yVAD-chloromethylketone) (Fig. S3A). Knockdown of NLRP3 orASC, but not NLRC4, in THP-1 macrophages caused a markedreduction of IL-1β release after LPS and Pam3CSK4 challenge(Fig. S3 B–D), suggesting the involvement of the NLRP3inflammasome in TLR2- and TLR4-induced IL-1β secretion.In addition, PAI-2 suppressed ASC oligomerization, anotherhallmark of inflammasome activation, in LPS-treated THP-1macrophages (Fig. 1D). The arginine residue at position 380of PAI-2 is crucial for its protease inhibition (16). THP-1macrophages expressing mutant PAI-2 containing an alaninesubstitution at residue 380 (A380) showed little inhibition inLPS-induced IL-1β and IL-18 production, caspase-1 activa-tion, and ASC oligomerization (Fig. 1 D and E), suggestingthat PAI-2 suppression of LPS-induced inflammasome acti-vation is dependent mainly on its protease inhibition function.For further evidence that PAI-2 modulates IL-1β maturationthrough NLRP3 activation, we coexpressed pro–IL-1β, pro-caspase-1, ASC, and NLRP3 in HEK293T cells to reconstitutethe NLRP3 inflammasome complex; the reconstitution ach-ieved was sufficient to allow autonomous secretion of matureIL-1β (Fig. 1F). Consistently, cotransfection of WT PAI-2, but notof the A380 mutant, blocked NLRP3-dependent pro–IL-1β pro-cessing. Collectively, these results demonstrated that TLR2 andTLR4 engagement up-regulates PAI-2 expression, which thennegatively regulates TLR-triggered activation of the NLRP3inflammasome.

PAI-2 Mediates Loss of Mitochondrial Integrity and a Decrease inMitochondrial ROS Production. ROS generation, lysosomal de-stabilization, and K+ efflux have been reported to be involvedin the activation of the NLRP3 inflammasome (1). To investigatethe underlying mechanism of PAI-2 suppression in LPS-stimulatedcells, we first examined whether ROS generation is involved inLPS-stimulated IL-1β maturation in PAI-2–deficient THP-1 mac-rophages. The addition of LPS to cells resulted in significant ROSgeneration (Fig. 2A and Fig. S4A). THP-1 macrophages treatedwith the antioxidant N-acetylcysteine, a ROS scavenger, potentlyinhibited LPS-induced caspase-1 activation and IL-1β productionbut not TNF production (Fig. S4B). Importantly, WT PAI-2, butnot the A380 mutant, significantly blocked LPS-triggered ROSgeneration in THP-1 macrophages (Fig. 2A and Fig. S4C). Mito-chondria are regarded as the major source of cellular ROS (18).Recent studies have reported that mROS promotes NLRP3 acti-vation (19). We found that LPS induces mROS generation inTHP-1 macrophages and that WT PAI-2, but not the A380 mu-tant, markedly blocks mROS production (Fig. 2B). Similarly,mROS generation increases significantly after LPS stimulation inPAI-2–knockdown BMDMs as compared with control cells (Fig.2C). mROS production has been shown to induce the relocation ofNLRP3 and ASC to mitochondria, leading to activation of theNLRP3 inflammasome (19). In support of this notion, LPS stim-ulation led to a marked increase in the colocalization of NLRP3and mitochondria in PAI-2–deficient macrophages, but PAI-2suppressed this effect (Fig. S4 D and E). Mitochondrial damageresulting in loss of mitochondrial membrane potential (ΔΨm) hasbeen linked to increased mROS production (19). Indeed, LPStreatment induced ΔΨm loss, and this effect was suppressed by

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Fig. 1. PAI-2 attenuates LPS-induced IL-1β production and caspase-1 activationin an NLRP3-dependent manner. (A) THP-1 cells were transduced with lentivi-ruses encoding the empty vector (control) or PAI-2-HA and were treated withLPS (100 ng/mL) for 8 h. (B and C) U937 cells (B) and BMDMs (C) were infectedwith lentiviruses encoding shRNA against luciferase (shLuc) or PAI-2 (shPAI-2)and were treated with LPS (100 ng/mL) for 4 and 24 h, respectively. (D) Controland PAI-2–expressing THP-1 macrophages were treated with LPS for 5 h. Celllysates were collected, and the ASC oligomers present in the lysates were pel-leted as described in Materials and Methods. (E) Control and THP-1 macro-phages expressing WT PAI-2 and the A380 mutant were treated with LPS (100ng/mL) for 8 h. (F) PAI-2 and NLRP3 were coexpressed as indicated with ASC,caspase-1 and pro–IL-1β in HEK293T cells for 36 h. Culture medium and celllysates were analyzed by immunoblotting using the indicated antibodies andELISA. The asterisk denotes a nonspecific band. All experiments were re-peated two or three times with similar results.

16080 | www.pnas.org/cgi/doi/10.1073/pnas.1306556110 Chuang et al.

WT PAI-2, but not by the A380 mutant, in THP-1 macrophages(Fig. 2D), suggesting that PAI-2 protects against LPS-inducedmitochondrial damage.We next determined the involvement of lysosomal destabilization

in LPS-stimulated IL-1β release. LPS stimulation in THP-1 mac-rophages resulted in a marked lysosome reduction until 2 h post-treatment, followed by recovery to the level of nontreatment at4 h posttreatment (Fig. S5A). In addition, active cathepsin B wasdetected in the supernatant of LPS-stimulated THP-1 macrophages(Fig. S5B). LPS-triggered IL-1β, but not TNF, release was abro-gated in cells pretreated with cathepsin B inhibitor, suggesting thatcathepsin B activity is required in TLR-triggered NLRP3 activation.Furthermore, suppression of ROS generation markedly blockedcathepsin B activation as well as IL-1β secretion in response to LPS(Fig. S5C). These data suggest that LPS induces ROS production,resulting in lysosomal destabilization and the activation of cathepsinB, which is required for IL-1β processing. Thus, PAI-2 expressionshould be able to abrogate lysosomal damage after LPS stimulation.Indeed, a significant reduction of LPS-stimulated loss of lysosomalintegrity was observed in THP-1 macrophages expressing PAI-2,whereas the A380 mutant had no effect on LPS-induced lysosomaldamage (Fig. 2E and Fig. S5D).K+ efflux is the third signaling pathway proposed to contribute

to the activation of the NLRP3 inflammasome. We found thatLPS-induced caspase-1 activation and IL-1β production, but notROS generation, were blunted in cells treated with extracellularKCl to inhibit K+ efflux (Fig. S5 E and F), suggesting that K+ effluxmediates LPS-induced IL-1β production in a ROS-independentmanner. In addition, K+ efflux was required for cathepsin Bactivation in LPS-activated THP-1 macrophages (Fig. S5G).Collectively, in PAI-2–depleted macrophages, LPS induces mROSgeneration, which, in combination with K+ efflux, results in

lysosomal destabilization/cathepsin B activation leading to pro–IL-1β processing.

PAI-2 Enhances Autophagy by Increasing the Level of the AutophagicProtein Beclin 1. Accumulating research has showed thatautophagy has a critical role in mROS production and NLRP3activation (8, 9, 19). To investigate whether PAI-2 regulatesautophagy, we detected the conversion of cytosolic LC3 (LC3-I)to the lipidated form of LC3 (LC3-II), which is enriched inautophagosome membranes. LPS engagement increased LC3processing, and PAI-2 expression further enhanced LPS-trig-gered LC3 conversion (Fig. 3A). In addition, PAI-2 also en-hanced the expression of the autophagic protein Beclin 1, but notof p62 (Fig. 3A and Fig. S6A), which previously was reported toregulate NLRP3 activation (20). Similar results were observed inPAI-2–knockdown BMDMs (Fig. 3B). To quantify autophagyinduction further, we monitored the number of autophagosomesin THP-1 macrophages exogenously expressing either EGFPfused to LC3 (EGFP-LC3) (21) or antibody-stained endogenousLC3. In line with previous reports, LPS increased EGFP-LC3puncta and endogenous LC3 lipidation in THP-1 macrophages(Fig. S6 B and C). PAI-2 significantly enhanced the number ofautophagosomes with or without LPS exposure, suggesting thatPAI-2 induces autophagy. In addition, the colocalization ofautophagy-associated p62 puncta with mitochondria was in-creased significantly in PAI-2 expressing macrophages upon LPStreatment (Fig. S6D), indicating that PAI-2 enhances mito-chondrial autophagy, probably for the purpose of removingdamaged mitochondria.Heat shock protein 90 (HSP90), a molecular chaperone,

recently was shown to regulate LPS-triggered autophagy throughassociation with Beclin 1 to protect Beclin 1 from proteasome-mediated degradation (21). We thus speculated that PAI-2 mightcomplex with HSP90/Beclin 1 to stabilize Beclin 1. PAI-2 indeedcoprecipitated with HSP90 and Beclin 1 when transientlyexpressed in HEK293T cells (Fig. S7 A and B). Likewise, Beclin1 was pulled down by PAI-2 (Fig. S7C). In addition, HSP90 as-sociated with endogenous Beclin 1 and PAI-2 in J774A.1 mac-rophages and BMDMs upon TLR4 engagement (Fig. 3C andFig. S7D). Consistent with previous results, geldanamycin (GA),an HSP90 inhibitor, promoted proteolytic degradation of Beclin1 in LPS-treated THP-1 macrophages; however, PAI-2 sup-pressed GA-induced Beclin 1 degradation (Fig. 3D). PAI-2 en-hanced the endogenous association of HSP90 and Beclin 1, andLPS stimulation further increased their interaction (Fig. 3E). InPAI-2–deficient THP-1 cells, the addition of the proteasomeinhibitor MG132 attenuated the reduction of Beclin 1 induced byLPS, but there was no observed effect of MG132 in cellsexpressing PAI-2 (Fig. S7E). Accordingly, PAI-2 blocked LPS-induced Lys48-linked ubiquitination of Beclin 1 (Fig. 3F). Inagreement with our finding, the PAI-2 A380 mutant lost itsability to bind HSP90 and Beclin 1 (Fig. 3G). Moreover, over-expression of Beclin 1 in PAI-2–deficient THP-1 macrophagesinhibited LPS-induced production of IL-1β and IL-18 and cas-pase-1 activation (Fig. 3H), confirming that PAI-2 inhibits LPS-triggered NLRP3 activation through Beclin 1. Collectively, theseresults demonstrate that PAI-2 associates with HSP90/Beclin 1to protect Beclin 1 from TLR4-triggered proteasome degrada-tion, resulting in autophagy induction.

PAI-2 Augments NLRP3 Degradation Through Autophagy. We no-ticed the NLRP3 protein level was reduced when cotransfectedwith PAI-2 in HEK293T cells (Fig. 1F), suggesting that PAI-2controls the expression of NLRP3 protein. Indeed, increasingamounts of PAI-2 protein caused a corresponding gradual de-cline in NLRP3 protein levels (Fig. S8A). PAI-2 did not alterLPS-activated NLRP3 mRNA levels (Fig. S2B), indicating thatPAI-2 regulates NLRP3 protein stability. To investigate thecellular mechanism for PAI-2’s modulation of NLRP3 proteinexpression, we examined the NLRP3 protein level in cellscotransfected with NALP3 and PAI-2 in the presence of

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Fig. 2. PAI-2 inversely regulates LPS-induced mitochondria dysfunction,mitochondrial ROS production, and lysosomal destabilization. (A) Controland THP-1 macrophages expressing WT PAI-2 or the A380 mutant weretreated with or without LPS (500 ng/mL) and then were stained with 5-(and6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Fluorescencewas measured with a microplate reader at the indicated time points. (B andC) Control and THP-1 macrophages expressing WT PAI-2 or the A380 mutant(B) or BMDMs infected with lentiviruses expressing shLuc or shPAI-2 (C) wereleft untreated or were treated with LPS (500 ng/mL) for 2 h and then werestained with MitoSOX followed by FACS analysis. (D) Control or PAI-2–expressing THP-1 macrophages were left untreated or were treated with LPS(500 ng/mL) for 2 h and then were stained with JC-1 dye followed by FACSanalysis. Data were assessed for the ratio of JC-1 fluorescence (red:green)and are presented relative to values in WT cells. (E) Control, WT PAI-2–, orA380mutant-expressing THP-1 macrophageswere treated with LPS (500 ng/mL)for the indicated times and then were stained with LysoTracker Red and wereanalyzed by FACS. Data shown are the fold-increase of fluorescence intensity instimulated cells relative to nonstimulated cells. All experiments were repeatedtwo or three times with similar results. *P < 0.05; **P < 0.01; ***P < 0.001.

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inhibitors that block two major protein degradation pathways.NLRP3 expression was increased markedly in cells treated withbafilomycin A1, which suppresses autophagosome maturation,whereas the proteasome inhibitor MG132 produced little changein the NLRP3 protein level (Fig. 4A), suggesting that PAI-2mediates NLRP3 degradation through the autophagy–lysosomepathway. Accordingly, in THP-1 macrophages expressing PAI-2or Beclin 1, LPS-stimulated NLRP3 expression was decreasedmarkedly (Figs. 3H and 4B). In contrast, depletion of PAI-2 inBMDMs resulted in increased NLRP3 protein levels after LPSchallenge (Fig. 4C). Furthermore, LPS enhanced the colocal-ization of NLRP3 and LC3-positive autophagosomes in PAI-2–expressing THP-1 macrophages, but this effect was barely visiblein PAI-2–deficient cells (Fig. 4D). Finally, suppression of NLRP3degradation by the autophagic inhibitors bafilomycin A1 or 3-methyladenine restored LPS-induced IL-1β maturation even inTHP-1 cells expressing PAI-2 (Fig. 4E and Fig. S8B). Collectively,these data demonstrate that, upon TLR4 activation, PAI-2 pro-motes NLRP3 degradation by enhancing autophagy, therebysuppressing NLRP3 activation and IL-1β maturation.

PAI-2 Attenuates E. coli-Induced IL-1β Secretion and Cell Death. Ithas been shown that nonpathogenic E. coli infection leads toNLRP3 activation and subsequent caspase-1–dependent IL-1β

production and pyroptosis (22, 23). To determine whether PAI-2could suppress NLRP3 activation induced by E. coli, we exam-ined IL-1β secretion and pyroptosis after E. coli challenge. UponE. coli infection, THP-1 macrophages expressing PAI-2 exhibiteda marked decrease in IL-1β release and cell death as judged byLDH release, as compared with the control cells (Fig. 5A).However, E. coli–induced TNF release was similar in both cells.Similar results were observed in E. coli–infected BMDMs inwhich PAI-2 was silenced (Fig. 5B). Accordingly, depletion ofPAI-2 induced a decrease in the level of Beclin 1 protein and anincrease in NLRP3 production upon E. coli challenge (Fig. S9 Aand B). These results confirm that PAI-2 is an important nega-tive mediator of the NLRP3 inflammasome-dependent IL-1βmaturation and cell death during E. coli infection.

DiscussionEmerging evidence has demonstrated that inflammasomes playa crucial role in the innate immune response, and dysregulationof their activities has been associated with pathogenesis of a va-riety of diseases (3). Thus, the mechanisms underlying the reg-ulation of inflammasomes have been investigated extensively.However, most studies have focused on the initiation or pro-motion of inflammasome assembly and activation (1, 2); theirnegative regulation remains largely unknown. Here, we identifya negative regulatory role of PAI-2 and its cellular mechanismsin modulating the activation of the NLRP3 inflammasome.Dual stimulation is required for full activation of NLRP3-

mediated IL-1β production in macrophages (8). One stimulus,such as TLR ligands, is required for priming cells to synthesizepro–IL-1β and NLRP3. A second stimulus, such as ATP, isnecessary for NLRP3 activation and subsequent IL-1β and IL-18maturation. A previous study showed that LPS alone is sufficientto induce caspase-1–dependent pro–IL-1β processing in IKKβ-deficient cells (15), suggesting an as-yet-unidentified, NF-κB-dependent negative regulatory mechanism for control ofinflammasome-mediated IL-1β production after TLR activation.In this study, we show that LPS alone is sufficient to induceNLRP3-dependent caspase-1 activation and IL-1β production inboth PAI-2–deficient THP-1 macrophages and PAI-2–silencedBMDMs or U937 macrophages. PAI-2 expression does not altergene expression at the transcriptional level after TLR stimula-tion. These results suggest that PAI-2 is a negative regulator ofTLR-mediated immune responses to control NLRP3 inflamma-some activation and IL-1β maturation.Cytosolic K+ efflux, ROS, and lysosomal rupture/cathepsin B

activation have been proposed to mediate NLRP3 activation bydifferent stimuli (1). In PAI-2–deficient THP-1 macrophages, wefound that LPS engagement results in ROS generation, sub-sequently causing lysosomal destabilization and cathepsin B ac-tivation, and that PAI-2 reverses these effects. How activecathepsin B triggers NLRP3 inflammasome activation remainspoorly understood despite years of effort. More recently, ca-thepsin B has been demonstrated to interact with NLRP3 anddrive its activation in chemotherapeutic agent-treated myeloid-derived suppressor cells in a proteolysis-independent manner(24). Our results, however, demonstrate that the proteolytic ac-tivity of cathepsin B is required for LPS-induced NLRP3 acti-vation, suggesting that cathepsin B may cleave an as-yet-unidentified mediator, driving activation of the NLRP3 inflam-masome. In addition, mROS, but not ROS derived fromNADPH oxidase, was shown to be pivotal for activation of theNLRP3 inflammasome (19). mROS accumulation can activateautophagy in mitochondria, possibly through reduction of theΔΨm. In turn, autophagy removes damaged mitochondria toavoid further mROS accumulation (19, 25). Thus, the inhibition ofautophagy causes a bulk of ROS production, resulting in NLRP3inflammasome activation. Similarly, our data show that loss ofPAI-2 leads to a decrease of autophagy and an increase of ΔΨ lossas well as mROS generation, which in turn results in NLRP3-dependent IL-1β production. Previously, TLR2/4 engagement wasshown to induce mROS generation, whereas endosomal TLR

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Fig. 3. PAI-2 induces autophagy through association with Beclin 1 andHSP90 and protects Beclin 1 from proteasome-mediated degradation. (A)Control and PAI-2–expressing THP-1 macrophages were stimulated with LPS(100 ng/mL) for the indicated times. (B) BMDMs infected with lentivirusesexpressing shLuc or shPAI-2 were stimulated with LPS (100 ng/mL) for 6 h. (C)J774A.1 macrophages were stimulated with LPS (500 ng/mL) for 4 h. (D)Control and PAI-2–expressing THP-1 macrophages were stimulated with LPS(100 ng/mL) for the indicated times in the absence or presence of 1 μMGA. (E)Control and HA-tagged PAI-2–expressing THP-1 macrophages were stimu-lated with LPS (500 ng/mL) for 6 h in the absence or presence of 1 μM GA. (F)Control and HA-tagged PAI-2–expressing THP-1 macrophages pretreatedwith MG132 (20 μM) were incubated with LPS (500 ng/mL) for 60 min. (G) HA-tagged WT PAI-2 or A380 mutant and Myc-tagged Beclin 1 were coex-pressed in HEK293T cells as indicated for 36 h. (H) Control and Beclin 1-expressing THP-1 macrophages were stimulated with LPS (100 ng/mL) for8 h. Protein levels of HSP90, Beclin 1, LC3, Lys48-linked Beclin 1, TNF, andIL-1β, and interactions between these proteins as indicated in each lanewere analyzed with immunoblotting or immunoprecipitation followedby immunoblotting. All experiments were repeated two or three timeswith similar results.

16082 | www.pnas.org/cgi/doi/10.1073/pnas.1306556110 Chuang et al.

ligands (TLR3 and TLR9) do not influence mROS production(26). It has been perplexing that stimulation with TLR2 and TLR4ligands alone is sufficient to induce mROS but fails to activate theNLRP3 inflammasome in macrophages. Our findings offer anexplanation for this puzzle, in that TLR2 and 4 ligands also inducePAI-2 expression, which enhances autophagy to eliminate thegenerated ROS to blunt NLRP3 activation.Autophagy was known to regulate NLRP3 inflammasome ac-

tivity negatively (8, 19), but the underlying mechanism remainedunknown. Our data unravel the mechanism by which autophagysuppresses NLRP3 activation by targeting NLRP3 protein forautophagy-lysosomal degradation. Autophagy is a cellular pro-cess that maintains cellular homeostasis by removing damagedproteins and organelles through lysosomes (7). Of note, NLRP3has been reported to be relocalized to mitochondria uponsensing mROS accumulation (19). Thus, the degradation ofNLRP3 in mitochondria through the autophagy–lysosomepathway during the process of PAI-2–enhanced autophagy for-mation would be expected. However, in addition to lowering thelevel of NLRP3 in the mitochondrial fraction, PAI-2 also de-creased cytosolic NLPR3 protein, suggesting that autophagy-

mediated NLRP3 degradation may not occur only in mito-chondria. Similarly, overexpression of the autophagic proteinBeclin 1 bypassed the requirement for PAI-2 in macrophagesand blunted NLRP3 activation by enhancing NLRP3 degrada-tion. Previously, NLRP3 protein degradation was shown to bedependent on the proteasome system (27). Our data indicatethat, in addition to the proteasome-dependent system, NLRP3 isdegraded primarily by the autophagy–lysosome pathway.In macrophages, signaling mediated by TLR ligands can trigger

autophagy, although the mechanism remains largely unknown (28,29). Mounting evidence indicates that the protein level of Beclin 1in cells is crucial for autophagy induction (30). HSP90 previouslywas shown to enhance LPS-induced autophagy by reducing Beclin1 degradation. HSP90 is an abundant molecular chaperone thatassociates with diverse proteins to stabilize its client proteins. Ourdata demonstrate that PAI-2 associates with Beclin 1 and HSP90to protect Beclin 1 from further ubiquitin-proteasome–dependentdegradation, thereby providing a molecular link for PAI-2 toenhance autophagy.PAI-2, a multifunctional protein, belongs to the SERPIN family,

which inhibits serine proteases (11). PAI-2 expression in mono-cytes and macrophages is up-regulated rapidly, mainly throughIKK/NF-κB activation, after stimulation with inflammatory medi-ators and pathogens (13). PAI-2 suppresses apoptosis and IL-1β–driven inflammation, although the underlying mechanisms arepoorly understood (13, 15). The present study provides the missinglink between IKK/NF-κB–mediated PAI-2 expression and the in-hibition of caspase-1–dependent IL-1β maturation: PAI-2 bindingwith HSP90/Beclin 1 protects Beclin 1 from proteosome degra-dation, resulting in enhanced autophagy as well as NLRP3 deg-radation (Fig. S9C). Interestingly, two viral serpin-like proteins—CrmA, a poxvirus serpin-like protease inhibitor, and Serp2, a 34-kDa serpin-like protein in myxoma virus (31)—can suppress theloss of ΔΨm and caspase-1 activation, resulting in a dampening ofthe host inflammatory response. Whether these two viral SERPIN-like proteins inhibit caspase-1 activity via a mechanism similar tothat of PAI-2 remains to be determined. In addition, our resultsdemonstrate that THP-1 macrophages expressing the PAI-2 A380mutant [a residue critical for its protease inhibition (16)] failed toinhibit LPS-induced IL-1β processing. PAI-2 A380 also lost itsbinding to HSP90/Beclin 1, resulting in loss of autophagy inductionin response to LPS. However, residue 380 of PAI-2 is crucial forretinoblastoma stability and protection from TNF-induced apo-ptosis (12, 32). Thus, whether residue 380 of PAI-2 is important inmaintaining only the HSP90/Beclin 1 complex or whether PAI-2

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Fig. 4. PAI-2 enhances NLRP3 degradation in an autophagy-dependentmanner. (A) HEK293T cells were cotransfected with PAI-2-HA and Flag-NLRP3. After 36 h, cells were treated with MG132 (20 μM) or bafilomycin A(100 nM) for 2 h. (B) Control and PAI-2–expressing THP-1 macrophages werestimulated with LPS (100 ng/mL) for the indicated times. (C) BMDMs infectedwith lentiviruses expressing shLuc or shPAI-2 were stimulated with LPS (100ng/mL) for 6 h in the absence or presence of bafilomycin A (100 nM). Celllysates were analyzed by immunoblotting using the indicated antibodies. (D)Control or PAI-2-HA–expressing THP-1 macrophages were incubated withLPS (500 ng/mL) and bafilomycin A (100 nM) for 6 h and were immunos-tained with antibodies against NLRP3 (red), LC3B (green), and HA (orange)followed by confocal microscopy. (Scale bars, 10 μm.) White arrows in theimages on the right correspond to the colocalization analysis of fluorescenceintensities measured by ZEN 2010 software. Colocalization coefficients ofNLRP3 and LC3B were quantified using Zeiss colocalization coefficient soft-ware and are shown in the lower panel (n = 10). (E) Control or PAI-2–expressing THP-1 macrophages were incubated with LPS (100 ng/mL) in thepresence or absence of bafilomycin A (100 nM) for 8 h. Culture medium andcell lysates were analyzed by immunoblotting using the indicated anti-bodies. All experiments were repeated two or three times with similarresults.

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Fig. 5. PAI-2 decreases IL-1β, but not TNF, production and cell death inresponse to E. coli infection. Control and PAI-2–expressing THP-1 macro-phages (A) or control and PAI-2–silenced BMDMs (B) were challenged withE. coli at a multiplicity of infection of 50. After infection for the indicatedtimes, culture supernatants were assayed for IL-1β and TNF by ELISA and forcell death by LDH release. Data are representative of two experiments donein triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

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binds to and inhibits subsequent proteases to protect Beclin 1 fromprotein degradation remains to be explored.The NLRP3 inflammasome is activated in response to a panel

of live bacteria including E. coli, Citrobacter rodentium, and Vibriocholerae (5). In addition, E. coli and C. rodentium are able toinduce caspase-1– and caspase-11–dependent cell death. Consis-tent with its role in the negative regulation of NLRP3 activationinduced by TLR ligands, we found that PAI-2 suppressed IL-1β,but not TNF, secretion in response to E. coli infection. In-terestingly, PAI-2 also protected against E. coli-induced cell deathin both human THP-1 macrophages and mouse BMDMs. Morerecent studies showed that caspase-11, rather than caspase-1,activation is crucial for E. coli-induced macrophage death (33,34). Caspase-4 and caspase-5 are assumed to be human homologsof mouse caspase-11, but their functional roles in E. coli-inducedcell death are still unknown. Thus, the mechanisms underlying thesuppression of E. coli-triggered pyroptosis by PAI-2 remain tobe elucidated.Collectively, our data show that PAI-2 induced by TLR4-

activated NF-κB associates with the HSP90/Beclin 1 complexand stabilizes Beclin 1 protein to enhance autophagy, result-ing in decreased mROS levels and increased NLRP3 proteindegradation, which in turn lead to reduced NLRP3 activation.Recently, the crosstalk between TLRs and the NLRP3 inflam-masome during the inflammatory response has been discussedintensively (33, 35). TLRs regulate the expression of pro–IL-1β,and the TLR–TRIF axis determines ROS generation and caspase-11 expression required for NLRP3 activation. Our findings notonly provide a mechanistic explanation for how IKK/NF-κB,

a core signaling component of the innate immune response, neg-atively modulates caspase-1–mediated IL-1β production but alsopresent PAI-2 as a negative regulator of NLRP3 activation fol-lowing TLR signaling, thereby tightly modulating IL-1β–driven inflammation.

Materials and MethodsMacrophage Isolation and Stimulation. Bone marrow was collected from fe-murs and tibia of C57BL/6 mice and used to generate BMDMs as describedpreviously (36). For the positive control of NALP3-dependent inflammasomeactivation, BMDMs were pretreated with LPS (500 ng/mL) for 3 h and pulsedwith ATP (5 mM) for 20 min as described in ref. 36.

Bacterial Infection. Early log-phase E. coli DH5α (Invitrogen) was added toTHP-1 macrophages and BMDMs at a multiplicity of infection of 50. Cultureplates were centrifuged at 190 × g for 5 min and incubated at 37 °C for60 min. The culture medium was replaced with fresh medium containinggentamicin (20 μg/mL) to kill extracellular bacteria, and infected cells werecultured for additional indicated times. shRNA target sequences are listed inTable S1, and primer sequences for RT-QPCR are listed in Table S2. Furtherexperimental details can be found in the SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Drs. J. Tschopp, F.-J. S. Lee, S. C. Lee, J.-T.Wu, C.-C. Chen, W.-W. Lin, and B. A. Wu-Hsieh for gifts of reagents, plasmids,and antibodies. Work in the L.-C.H. and T.-H.C. laboratories was supportedby National Science Council (NSC) Grants NSC-98-2628-B-002-034 (to L.-C.H.)and NSC-100-2320-B-400-011 (to T.-H.C.) and by Grant NHRI-EX102-10257SIfrom the National Health Research Institutes, Taiwan. RNAi reagents wereobtained from the National RNAi Core Facility supported by the NationalCore Facility Program for Biotechnology Grants of the NSC.

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16084 | www.pnas.org/cgi/doi/10.1073/pnas.1306556110 Chuang et al.